The invention relates generally to the fields of enzymology and nucleic acid analogs. Specifically, this invention is directed to template-dependent ligation of PNA-DNA chimeras and oligonucleotides with ligase enzymes.
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The covalent joining of nucleic acid probes by ligase enzymes is one of the most useful tools available to molecular biologists. When two probes are annealed to a template nucleic acid where the two probes are adjacent and without intervening gaps, a phosphodiester bond can be formed by a ligase enzyme (Whiteley, 1989). The ligation bond is formed between a 5xe2x80x2 terminus of one probe and the 3xe2x80x2 terminus of the other probe.
The events of annealing and ligation each require a high level of fidelity, i.e. complementarity, between the sequences of the ligating probes and the template nucleic acid. Both events are inefficient when base-pairing mismatches occur. Generally, DNA ligase can join two adjacent probes only when they perfectly complement a denatured template nucleic acid, such as a PCR product (Landegren, 1988; Nickerson, 1990). Even a single nucleotide mismatch at, or near, the ligation site of the probes will prevent ligation of the annealed probes.
Oligonucleotide ligation assays detect the presence of specific sequences in target DNA sample. For example, allelic discrimination assays rely on probes representing the complementary sequences of the allelic forms to the target. Ligation to a common, second target-complementary probe indicates the presence of the polymorphic site (Whiteley, 1989; Landegren, 1988). Absence of ligation indicates the lack of the polymorphic site. Ligation can be detected through detectable labels on the allelic probe and electrophoretic separation of the ligation products (Grossman, 1994).
It is desirable to provide optimized probes and methods of annealing and ligation. Such methods would improve assays and tests that benefit from greater precision and accuracy.
The invention relates to chimeric molecules of PNA and DNA monomer units and their use in ligation methods to generate ligation products. The invention is based in part on the discovery that a ligase enzyme can ligate a PNA-DNA chimeric probe and a second probe under a broad range of experimental conditions and variables. PNA-DNA chimeras of the invention comprise at least two moieties covalently linked together, preferably: i) a contiguous moiety of 3 to 15 PNA monomer units, and ii) a contiguous moiety of at least two nucleotides. The nucleotide moiety has a ligatable terminus, such that the PNA-DNA chimera can be ligated to a second probe.
In a first aspect, the invention provides a method of producing a template-dependent ligation product by ligating a PNA-DNA chimeric probe, annealed to a template nucleic acid in the presence of a ligase and a ligation reagent, to a second probe annealed adjacent to the chimeric probe on the template nucleic acid. The second probe is capable of supporting template-dependent ligation. The second probe is a PNA-DNA chimera or an oligonucleotide. The second probe may be 5 to 100 monomer units or nucleotides (nt) in length. Preferably the second probe is 10 to 30 nt. Together, the chimeric probe and the second probe may be 10 to 100 nt.
In one illustrative embodiment of the invention, the PNA-DNA chimera has the formula:
Px-L-Ny
where each P is independently a PNA monomer, x is an integer from 3 to 15, L represents a covalent linkage between P and N, each N is independently a nucleotide, y is an integer from 2 to 15, and the terminal N has either a 3xe2x80x2 hydroxyl group or 5xe2x80x2 hydroxyl group.
In a preferred embodiment, the PNA moiety, i.e., Px, of the PNA-DNA chimera is a 2-aminoethylglycine peptide nucleic acid.
The DNA moiety, i.e., Ny, of the PNA-DNA chimera may be comprised of 2xe2x80x2-deoxynucleotides (DNA), ribonucleotides (RNA), and modified sugars or internucleotide linkages thereof, especially those that confer greater specificity, affinity, rate of hybridization, and chemical stability.
The chimera and/or the second probe may be labelled with a non-radioisotopic label such that the ligation product is non-radioisotopically labelled. In embodiments employing a labelled PNA-DNA chimera, the PNA-DNA chimera may be labelled at: (i) a nucleobase, e.g. the 7-deaza or C-8 positions of a purine or a deazapurine nucleobase, or the C-5 position of a pyrimidine nucleobase; (ii) a sugar; (iii) the PNA backbone; or (iv) an amino, a sulfide, a hydroxyl, and/or a carboxyl group. Preferably, the chimera is labelled at the amino terminus of the PNA moiety. In embodiments employing a labelled oligonucleotide, the oligonucleotide is preferably labelled at the opposite terminus from the ligation site, 3xe2x80x2 or 5xe2x80x2. Alternatively, the oligonucleotide may be labelled at a nucleobase, but may also be labelled at other positions provided that the label does not interfere adversely with hybridization affinity or specificity, or with ligase efficiency. Labels may be fluorescent dyes, fluorescence quenchers, hybridization-stabilizers, energy-transfer dye pairs, electrophoretic mobility modifiers, chemiluminescent dyes, amino acids, proteins, peptides, enzymes, and affinity ligands. Preferably, the label is detectable upon illumination with light, e.g. laser sources at infrared, visible or ultraviolet excitation wavelengths.
The PNA and DNA moieties of the chimeric probe are covalently linked together. The linkage, L, between the PNA and DNA moieties may be a bond, e.g. the carbonyl-nitrogen bond in an amide group where the moieties are linked without intervening atoms, or a multi-atom linker. The linkage may comprise a phosphodiester group or a phosphoramidate group.
The template or target nucleic acid can be any nucleic acid or nucleic acid analog capable of mediating template-directed nucleic acid synthesis. Examples of suitable template nucleic acids include, e.g., genomic DNA, DNA digests, DNA fragments, DNA transcripts, plasmids, vectors, viral DNA, PCR products, RNA, and synthetic nucleic acids. The template nucleic acid may also be a metaphase or interphase chromosome. Preferably, the chromosome is denatured prior to PNA-DNA chimera hybridization and ligation. Template nucleic acids may be single-stranded or double-stranded and can range from as few as about 20-30 to as many as millions of nucleotides (nt) or base-pairs (bp), depending on the particular application.
The template nucleic acid, the PNA-DNA chimera, or the second probe may be immobilized on a solid substrate. Ligations may be conducted where one of the probes or template is attached to a solid support or surface.